Mitochondrial myopathy with mitochondrial DNA (mtDNA) depletion is an important and newly recognized class of mitochondrial disease that causes metabolic myopathy as well as intellectual and developmental disabilities. Deficiency of ADP-specific succinyl-CoA synthetase (SCS), a component of the TCA cycle, has been identified as one of the causes of mitochondrial myopathy with mtDNA depletion. A gene trap mutant clone of Sucla2, the gene encoding the beta-subunit of SCS, has been isolated. This mutant mouse ES cell line has been used to generate transgenic mice and mouse embryonic fibroblast (MEF) cell lines deficient for Sucla2. Mutant MEFs as well as mutant embryonic skeletal muscle and brain demonstrate mtDNA depletion. This proposal is based on the hypothesis that SCS is an important protein component of the mtDNA nucleoid complex required for mtDNA maintenance and stability. Cells deficient for SUCLA2 will be utilized to investigate the functional and structural components of SUCLA2 required for mtDNA maintenance. Genetic studies to determine proteins that interact with SCS and are potential components of the mtDNA nucleoid complex will also be pursued. Mice mutant for Sucla2 will be generated to study the pathophysiology of SCS deficiency as a model for mtDNA depletion and myopathy. These studies will provide new insights into fundamental mechanisms of mtDNA biology as well as provide a novel model of mtDNA depletion to facilitate the development of new therapeutic strategies for an important subset of metabolic myopathies.